Bacteria

Bacteria play a central role in genetic engineering because they are easy to grow, simple to manipulate, and naturally exchange genetic material. In this chapter we focus specifically on bacterial features that are important for genetic engineering, not on their general biology or their role as pathogens.

Why Bacteria Are Ideal Tools in Genetic Engineering

Several properties make bacteria especially useful in molecular biology and biotechnology:

They grow very fast (typical generation time for E. coli under good conditions: about 20 minutes).
They are easy and inexpensive to cultivate in large numbers.
Their cells are structurally simple compared with eukaryotic cells.
Their genomes are relatively small and mostly well understood.
They naturally possess mechanisms for taking up, exchanging, and rearranging DNA, which can be harnessed or imitated in the lab.

Because of these properties, bacteria are widely used as:

Cloning hosts (for amplifying DNA fragments).
Expression systems (for producing proteins, e.g. insulin).
Model organisms (for studying gene regulation, mutation, and recombination).

Most basic genetic engineering methods were first developed in, and are still often demonstrated with, the bacterium Escherichia coli (E. coli).

Bacterial Genetic Material Relevant to Genetic Engineering

Bacterial Chromosome

Most bacteria have:

A single, typically circular DNA molecule called the bacterial chromosome.
DNA that is not surrounded by a nuclear membrane (prokaryotic cell organization).
Genes that are often arranged in operons (clusters of genes transcribed together), which is important for understanding and manipulating gene expression in bacteria but will be discussed in detail elsewhere.

For genetic engineering, the bacterial chromosome can sometimes be directly altered (e.g. by gene knockouts or insertions), but more commonly, foreign DNA is maintained on plasmids.

Plasmids

Plasmids are small, usually circular DNA molecules that:

Exist independently of the bacterial chromosome.
Replicate on their own (they have their own origin of replication).
Can carry genes that provide special functions (e.g. antibiotic resistance, metabolic capabilities, virulence factors).
Can be transferred between bacteria, sometimes even between different species.

In genetic engineering, plasmids are:

Modified to create cloning vectors and expression vectors.
Used as carriers (vehicles) for foreign DNA.

Key plasmid features important for genetic engineering include:

Origin of replication (ori)
Determines:

Whether the plasmid can replicate in a particular bacterial species.
The copy number (how many copies per cell).
High-copy plasmids yield lots of DNA and often high protein expression; low-copy plasmids can be useful when high expression is toxic to cells.

Selectable marker
Usually a gene that confers resistance to a specific antibiotic (e.g. ampicillin, kanamycin, chloramphenicol).
Only bacteria that have taken up the plasmid can grow on medium containing that antibiotic.
Multiple cloning site (MCS, polylinker)
A short region containing many unique restriction enzyme recognition sites.
This is where foreign DNA fragments are typically inserted.
Promoters and regulatory elements (mainly in expression vectors)
Control transcription of the inserted gene.
Often, inducible promoters are used, which can be turned on or off by adding specific molecules (e.g. IPTG for the lac-derived promoters).

Plasmids occur naturally in many bacteria, but most plasmids used in labs are artificially constructed from natural plasmids and engineered DNA sequences.

Natural Mechanisms of Gene Transfer in Bacteria

For genetic engineering, it is important to understand how bacteria naturally exchange genetic material, because many lab techniques are based on or inspired by these processes.

Transformation

Transformation is the uptake of free DNA from the environment:

DNA is released when cells die and break apart.
Some bacteria are naturally competent: they have specialized systems for binding and transporting DNA across their cell envelope.
Once inside, the DNA fragment can recombine with the bacterial chromosome or exist as a plasmid (if it contains the necessary sequences).

In genetic engineering:

Transformation is mimicked or induced artificially.
Bacteria like E. coli are made competent in the lab (e.g. by treatment with CaCl₂ or by electroporation) to take up plasmid DNA.

Conjugation

Conjugation is the direct transfer of DNA from one bacterial cell to another:

It often involves F plasmids (fertility plasmids) or other conjugative plasmids.
A pilus (a tubular appendage) is formed between a donor and a recipient cell.
A copy of the plasmid DNA is transferred through the pilus.

For genetic engineering:

Conjugative plasmids (or plasmids with conjugation functions) can be used to deliver DNA into bacteria that are hard to transform.
Conjugation can transfer large DNA segments or plasmids between different bacterial strains and sometimes species.

Transduction

Transduction is the transfer of bacterial DNA by bacteriophages (bacterial viruses):

In generalized transduction, phages accidentally package fragments of bacterial DNA and deliver them to another bacterium during infection.
In specialized transduction, temperate phages excise incorrectly from the chromosome and carry nearby bacterial genes.

Although transduction is a natural process, in genetic engineering:

Phage-derived systems and concepts are adapted for specialized applications (e.g. phage-based cloning systems, certain genome editing tools).
Transduction can be used to move specific chromosomal alleles between bacterial strains in research labs.

Laboratory Introduction of DNA into Bacteria

While natural transformation, conjugation, and transduction occur in nature, genetic engineering uses controlled lab methods to introduce defined DNA constructs into bacterial cells.

Chemical Transformation

In chemical transformation:

Bacteria are treated with solutions (commonly CaCl₂) to become competent.
Plasmid DNA is mixed with these cells.
A short heat shock (e.g. 42 °C for ~30–60 seconds) creates conditions that allow DNA to enter cells.
After recovery in a nutrient medium, cells are spread onto agar plates containing an antibiotic; only cells that have taken up plasmid survive.

Key points:

Simple and inexpensive.
Often yields a moderate number of transformants.
Widely used for routine plasmid cloning in E. coli.

Electroporation

Electroporation uses an electric field to make bacterial membranes temporarily permeable:

Bacteria and DNA are mixed in a conductive solution.
A short, high-voltage pulse is applied in a special cuvette.
The electric pulse forms transient pores in the bacterial membrane through which DNA enters.
Cells are then allowed to recover and are plated on selective medium.

Key points:

Usually produces more transformants than chemical methods.
Can be used for many bacterial species, including ones that are difficult to transform chemically.
Requires specialized equipment (electroporator, special cuvettes).

Conjugation-Based DNA Transfer in the Lab

Conjugation can be exploited to introduce plasmids into bacteria:

A donor strain carries a conjugative plasmid or helper plasmid.
A recipient strain lacks the plasmid but has some selectable trait (e.g. resistance to another antibiotic).
Donor and recipient are mixed, incubated together, and then plated on medium selecting for both plasmid and recipient traits.

This is especially useful for:

Bacteria that cannot be efficiently transformed or electroporated.
The transfer of large plasmids that are difficult to handle by other methods.

Selection and Screening of Bacterial Transformants

After introducing DNA into bacteria, not all cells will have taken up the desired DNA. Two concepts are crucial:

Selection: Ensuring that only bacteria with the plasmid (or chromosomal modification) can grow.
Screening: Distinguishing between cells that have the correct DNA construct and those that have not.

Antibiotic Selection

Most plasmid vectors carry an antibiotic resistance gene:

Medium contains the corresponding antibiotic.
Only bacteria that carry the plasmid survive and form colonies.

This step identifies bacteria that have any plasmid, but not necessarily the plasmid with the correct insert.

Screening for Correct Inserts

Several strategies are used to detect whether the foreign DNA fragment is correctly inserted:

Blue–white screening (involving the lacZ gene and a chromogenic substrate, often used in E. coli).
Colony PCR: small parts of colonies are used as templates for PCR to test for the presence and size of the insert.
Restriction digestion analysis of isolated plasmid DNA.
Sequencing of the insert region for precise confirmation.

These screening techniques are tightly linked to how bacteria handle plasmids and how plasmid vectors are designed.

Bacteria as Protein Production Factories

Once bacteria carry an expression plasmid with a foreign gene, they can be used to produce the corresponding protein.

Expression Vectors in Bacteria

Expression plasmids usually contain:

A strong bacterial promoter upstream of the foreign gene.
A ribosome-binding site for efficient translation initiation.
Often an inducible system that allows control over when and how much protein is produced (e.g. using an inducer like IPTG).
Sometimes fusion tags (e.g. His-tag) to simplify protein purification with chromatography.

Because bacteria grow quickly and reach high densities, they can produce large amounts of recombinant proteins, which are then purified for:

Medical use (e.g. human insulin, growth hormone).
Industrial enzymes.
Research purposes.

Limitations of Bacterial Expression

While bacteria are powerful production systems, they have some limitations:

They often cannot correctly process certain eukaryotic proteins that require specific post-translational modifications (e.g. glycosylation).
Very complex or membrane-bound eukaryotic proteins may fold incorrectly or aggregate.

These limitations are reasons why other expression systems (yeast, insect cells, mammalian cells) are also used in biotechnology. However, for many applications, bacteria remain the simplest and most economical choice.

Genetically Modified Bacteria in Research and Biotechnology

Bacteria are modified genetically for various purposes:

Model systems:

To study gene regulation, mutation, and DNA repair.
To dissect metabolic pathways by creating gene knockouts or overexpressing certain genes.

Biotechnological production:

Manufacturing amino acids, vitamins, antibiotics, and other valuable substances.
Producing recombinant vaccines and therapeutics.

Environmental applications (bioremediation):

Engineering bacteria to degrade pollutants or convert waste products.

Synthetic biology:

Constructing bacteria with completely new metabolic capabilities.
Designing genetic circuits that respond to environmental signals.

Because bacteria can multiply and spread rapidly, the use of genetically modified bacteria is closely regulated and typically confined to controlled environments. Containment strategies include:

Using strains that are weakened and cannot survive well outside the lab.
Designing plasmids and constructs that reduce the chance of uncontrolled spread of engineered genes.

Bacterial Defense Systems and Genetic Engineering

Bacteria themselves have evolved systems to protect against foreign DNA (especially phages). Understanding these systems has led directly to key tools in genetic engineering.

Restriction–Modification Systems

Many bacteria possess restriction–modification systems:

Restriction enzymes (restriction endonucleases):

Recognize specific short DNA sequences.
Cut DNA at or near these sequences.
This defends the bacterium against foreign DNA that lacks the appropriate protective modifications.

Modification enzymes (usually methyltransferases):

Methylate the bacterium’s own DNA at specific sites.
Protect it from cleavage by the corresponding restriction enzyme.

Genetic engineering harnesses restriction enzymes to cut and join DNA molecules at defined sequences, a topic covered in detail elsewhere. These enzymes were originally discovered and characterized in bacteria.

CRISPR–Cas Systems (Overview in Bacterial Context)

Many bacteria and archaea also possess CRISPR–Cas systems:

Function as an adaptive immune system against viruses and plasmids.
Store short sequences from invading DNA between repetitive elements in the genome (the CRISPR array).
Use RNA derived from these sequences to guide Cas proteins to recognize and cut matching foreign DNA during subsequent infections.

Although the molecular details belong to more specialized chapters, the important point here is:

CRISPR–Cas systems are natural bacterial defense mechanisms.
These systems have been adapted into powerful, programmable tools for genome editing in many organisms.

Summary

For genetic engineering, bacteria are central because they:

Provide natural DNA transfer mechanisms (transformation, conjugation, transduction).
Contain elements such as plasmids and restriction–modification systems that have been turned into essential molecular tools.
Serve as flexible, efficient hosts for DNA cloning and protein production.
Offer a simple, well-understood platform for designing, testing, and applying genetic modifications in both basic research and biotechnology.

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